Investigation of memory-enhancing effects of Streptococcus thermophilus EG007 in mice and elucidating molecular and metagenomic characteristics using nanopore sequencing

Over the past decades, accumulating evidences have highlighted the gut microbiota as a key player in the brain functioning via microbiota–gut–brain axis, and accordingly, the beneficial role of several probiotic strains in cognitive ability also have been actively investigated. However, the majority of the research have demonstrated the effects against age-related cognitive decline or neurological disease. To this end, we aimed to investigate lactic acid bacteria strains having beneficial effects on the cognitive function of healthy young mice and elucidate underlying characteristics by carrying out nanopore sequencing-based genomics and metagenomics analysis. 8-week consumption of Streptococcus thermophilus EG007 demonstrated marked enhancements in behavior tests assessing short-term spatial and non-spatial learning and memory. It was revealed that EG007 possessed genes encoding various metabolites beneficial for a health condition in many aspects, including gamma-aminobutyric acid producing system, a neurotransmitter associated with mood and stress response. Also, by utilizing 16S–23S rRNA operon as a taxonomic marker, we identified more accurate species-level compositional changes in gut microbiota, which was increase of certain species, previously reported to have associations with mental health or down-regulation of inflammation or infection-related species. Moreover, correlation analysis revealed that the EG007-mediated altered microbiota had a significant correlation with the memory traits.


Results and discussion
Effects of S. thermophilus EG007 and L. plantarum A003F7 on learning and memory of young healthy mice. General effects of probiotics. Each mouse has consumed an average of 1.08 × 10 10 CFU of S. thermophilus EG007 (ST group) or 3.12 × 10 10 CFU of L. plantarum A003F7 (LP group) per day throughout the whole study (8 weeks) (Supplementary Fig. 1). The average body weight during the course of the experiment is shown in Supplementary Fig. 1. The curve of body weight gain for the LP group is constantly under two other groups on the graph, but there was no significant difference between treatments for the total body weight gain (week0-week8) (one-way ANOVA, P = 0.494). Also, we did not observe any abnormal behavior nor subject mortality during the probiotic supplementation period. Thus, we could conclude that the consumption of both probiotics had no adverse effect on overall health. Y-maze spontaneous alternation test. The effects of EG007 and A003F7 on the short-term spatial working memory and exploratory behavior enhancement were evaluated using Y-maze spontaneous alternation after 5 weeks of administration. In this test, mice were allowed to freely explore three arms, and the number of total arm entries and alternation score were calculated. In the spontaneous alternation score, both LP and ST groups showed the enhanced ability to discriminate between maze arms compared with the control group. As shown in Fig. 2, a significant effect of treatment on the alternation score was observed (67.4 ± 2.72% vs. 65 ± 5.7% vs. 47.7 ± 4.93% for ST, LP, and Control, Kruskal-Wallis, P = 0.00956), with post-hoc analysis revealing that the ST group exhibited significantly improved short-term spatial memory (Tukey, P = 0.0354). We did not observe any significant differences between LP mice and control mice. The number of total arm entries was similar across all experimental groups, showing no difference in the general locomotor activity. And there was no correlation between the number of total arm entries and the alternation score (Pearson's correlation, r = 0.16, P = 0.18), indicating that any differences in locomotor activity did not influence the alternation score (Fig. 2). These results suggest that the administration of S. thermophilus can improve spatial learning and memory in healthy young mice.
Y-maze forced alternation test. After 7 weeks of administration, the forced alternation test was performed. For latency before entering the first novel arm (Fig. 2), the control group showed delayed entry into the novel arm, compared to the probiotics-treated groups, although the differences were not statistically significant (47 ± 19.6 s vs. 46.6 ± 9.94 s vs. 65.2 ± 20 s for ST, LP, and Control, Kruskal-Wallis, P = 0.7761). Regarding the percent time spent in the novel arm over the other arms, both ST mice and LP mice showed a higher percentage than the control group (29 ± 5.76% vs. 31.6 ± 9.22% vs. 23.1 ± 6.06% for ST, LP, and Control), but there were no statistically significant differences (one-way ANOVA, P = 0.666). Consistent with this, the percentage of entries into the novel arm over the other arms for both probiotics-fed groups were higher compared to that for the control group, with no significant differences between the groups (44.4 ± 3.29% vs. 45.2 ± 9.64% vs. 37.7 ± 7.89% for ST, LP, and control, Kruskal-Wallis, P = 0.9913).
Novel object recognition test. The novel object recognition test was conducted to test short-term nonspatial working memory in mice after 6 weeks of probiotics administration. In this test, the memory performance was assessed by measuring the discrimination ratio. When comparing the total time of investigation of both objects,  1.08 x 10 CFU of S. thermophilus EG007 3.12 x 10 CFU of L. plantarum A003F7 Control (water) Experiment Age w4 w5 w9 w10 w11 w12 10 10 Figure 1. Schematic diagram of study design. Male SPF C57BL/C mice (n = 36) were randomly divided into three groups, and each group was supplemented with S. thermophilus EG007, L. plantarum A003F7, and water (control). From week 5 (nine weeks of age) onward, all mice were subjected to the same series of behavior tests to assess their cognitive performance, the Y-maze test (spontaneous alternation and forced alternation) for spatial memory, the novel object recognition test for nonspatial memory, and the passive avoidance test for longterm associative memory. After 1 day following the last test, fecal samples were collected for gut microbiome community analysis, and all mice were sacrificed after 5 days.     Fig. 2). There was a significant difference between groups (one-way ANOVA, P = 0.03), and post-hoc analysis indicated that only ST mice had significantly increased preference for the novel (Tukey, P = 0.0264). These results indicated that the administration of S. thermophilus can help enhancing the ability to discriminate between familiar and novel objects through improving nonspatial memory skills in healthy C57BL mice.
Passive avoidance test. After 8 weeks of administration, the fear-motivated passive avoidance test was performed to evaluate the long-term associative memory of mice. In this test, memory performance was indicated by the latency to enter the dark chamber where the moue had experienced a foot shock. Therefore, more increased latency indicates better memory retention. As shown in Fig. 2 Control, respectively), indicating the formation of the contextual memory, but no significant differences were shown between the groups (Kruskal-Wallis, P = 0.969).
In summary, a series of behavior tests assessing different aspects of cognitive skills indicated EG007 significantly increased short-term spatial (SA) and non-spatial (NOR) learning and memory in healthy young C57BL/C mice. So far, the influence of S. thermophilus on the host CNS function has only been investigated in forms of probiotics mixture or yogurt in few studies 53,54 , and this is the first study to examine the effects of a single S. thermophilus strain. Although previous studies observed some L. plantarum strains were capable of relieving opposition/defiance behaviors in children with ASD 55 or attenuating cognitive impairments in the AD mouse model 56 , A003F7 did not show significant enhancements in this study.
Constructing complete genome of S. thermophilus EG007 and characterization of general genome features. In order to determine the genomic characteristics of EG007, we constructed the complete genome of S. thermophilus EG007. Flongle sequencing using 20 K insert size library generated 106,853 raw reads, totaling approximately 1.0Gbp of sequences (529.14x coverage) ( Table 1). After removing adapter sequences and chimeric reads using Porechop, reads with a quality score below 10 were discarded using Nanofilt, and 77,148 reads (a total of 737.7 Mb) remained. Reads had an average length of 9562.56 bp and N50 length of 14,953 bp, and they were subjected to genome assembly using CANU. As shown in Table 1, CANU successfully generated 2 contigs, with a total length of 1,901,104 bp, including contigs of 1,894,187 and 6917 bp. In order to reduce insertion/deletion errors which could affect the gene prediction, multiple rounds of polishing with racon and medaka were conducted until no additional correction could be made. Polishing steps reduced the number of pseudogenes from 435 to 218 and resulted two contigs of 1,895,271 bp and 6932 bp, and the smaller contig was confirmed as a plasmid using Plasflow. Lastly, the assembled reads were circularized using Circlator, and the completed genome of EG007 consisted of a circular chromosome 1,860,782 bp in size and a 3500 bp plasmid with 39.05% of GC contents, which were in range with other available S. thermophilus strains.
Prokka annotation indicated that the genome contained 2118 genes (CDS: 1952, misc_RNA: 80, rRNA: 18, tRNA: 67, tmRNA: 1) including two genes (replication protein and type 1 restriction enzyme protein) located in the plasmid. The general genomic structure of the EG007 is depicted in Supplementary Fig. 2. The predicted CDSs were then assigned in clusters of orthologous group (COG) functional categories ( Supplementary Fig. 3). 1554 proteins were classified into 19 specific COG categories, and the majority of them were involved primarily in housekeeping and metabolic processes. Among them, translation, ribosomal structure and biogenesis (J) and DNA replication, recombination and repair (L) represented the two most abundant categories. Also, an inspection of gene contents in metabolism revealed that EG007 seemed biased towards four categories: amino acid (E), inorganic ion (P), carbohydrate (G), and nucleotide (F) transport and metabolism, which represents 32% of assigned proteins.
Analysis using resfinder, CARD-rgi, and VFDB indicated that EG007 neither contain any antibacterial resistance genes nor virulence-related genes, whereas some strains of S. thermophilus were reported to possess resistance genes to tetracycline or erythromycin 57 . Also, 13 genomic islands (named from GI 1 to GI 13) (Supplementary Fig. 2 58 . In addition, CRISPRCasFinder detected three distinct CRISPR/Cas loci (CRISPR1, CRISPR2, and CRISPR3), similar to other S. thermophilus strains 59 . CRISPR-Cas system is widely distributed among prokaryotes as an adaptive immune system against bacteriophage infection. However, CRISPR1 of EG007  60 . This suggests a possible effective defense mechanism of the CRISPR1 against different bacteriophages compared to those of other sequenced S. thermophilus strains, thereby conferring a better adaptive immunity. Consistent with these, five incomplete prophage-like regions were identified in the genome which probably are traces of the ancient phage infections, followed by inactivation and slow decay 61 , indicating no recent infection occurred.
Identification of genes associated with psychobiotics potential in S. thermophilus EG007. We further investigated the functionality of the strain as probiotics and potential psychobiotics. First, for bacterial strains to be effective as probiotics, the ability to survive through the gastrointestinal (GI) tract environment is a key factor. Genomic analysis of EG007 showed that it presented coding sequences that possibly confer resistance abilities to various stress. A total of 24 genes that lead to acid tolerance (9 genes), Oxidative stress tolerance (11 genes), and bile salt tolerance (1 gene) were detected in the genome (Supplementary Table 1). Three genes associated with strain's adhering ability were also found, which was an important feature for successful colonization in the GI tract after intake. In addition, the genome showed the presence of extracellular polymeric substance (EPS) genes cluster which was involved in the extracellular polysaccharide synthesis (Supplementary Table 1). The presence of EPS genes gives probiotics important industrial properties related to the desired texture or reduced syneresis of fermented dairy products and health-promoting properties such as antimicrobial activity, anti-inflammatory activity, and immunomodulation 62,63 . Moreover, it was also studied that the EPS synthesis correlated with robustness in cell aggregation, and thereby providing higher survival properties in the GI tracts 64 . The structural and compositional diversity of EPS molecules are varied by both number and the type of genes organized in the clusters, and many different kinds of clusters have been discovered in S. thermophilus. The current analysis revealed a relatively large size (28,057 bp) of the cluster in EG007 which were composed of 33 genes (from deoD to permease gene) including UDP-galactopyranose mutase gene, rarely found in S. thermophilus EPS clusters 65,66 , and interestingly, it contained several hypothetical genes and transposase genes distributed downstream in the cluster. These observations were also reported in strain JIM8232, CNRZ368, and ND03. The transposase genes were located in the GI 6. Lastly, screening of the genome using BAGEL4 revealed a total of five putative bacteriocin biosynthesis gene clusters (cluster1: streptide; cluster2: sactipeptides; cluster3: Blp family; cluster4: lanthipeptides class 1; cluster5: sactipeptides), together with genes coding for the self-immunity protein and the transport system ( Supplementary Fig. 4, Supplementary Table 1). Bacteriocin-producing ability of probiotics is a key feature as these antimicrobial peptides can facilitate competition of the probiotics by directly eliminating pathogens and also serve as signaling peptides 67 . Similar to the genes in the EPS cluster, all five clusters were carried by genomic islands (cluster 1 in GI 10; cluster 2 in GI 5; cluster 3 in GI 11; cluster 4 in GI 2; cluster 5 in GI 13), and the lantibiotic biosynthesis genes (in cluster 4) showed high similarity with Streptpcoccus dysgalactiae (100% coverage, 97.18% identity) and Streptococcus pneumoniae (98% coverage, 80.28% identity).
These results indicate that there might have been horizontal gene transfers (HGT) events of EPS and bacteriocin clusters, and it provided EG007 beneficial traits for improved adaptability in the GI environment and probiotic functionalities. It is well known that a large proportion of neurotransmitters such as serotonin, dopamine, and GABA is produced in the gut 68 . Several studies confirmed that lactic acid bacteria were one of the most important neurotransmitter producers among the enteric microbes, and some of S. thermophilus strains with GABA-producing ability have also been discovered. GABA is a bioactive molecule that exerts several beneficial physiological functions in humans and animals 69 . The biosynthesis of GABA by enteric microbes is performed by the glutamate decarboxylase (GAD) system, which is composed of the GAD enzyme (encoded by gadA or gadB) and glutamate/GABA antiporter GadC 70 . Once GadC transports l-glutamate into a cell, it is decarboxylated by GAD enzyme to form GABA 70 , and the decarboxylated product GABA is exported back to the extracellular matrix through GadC 70 . Generally, the microorganisms with the GAD gene are known to be capable of synthesizing GABA 71 . Genome analysis of EG007 revealed that the strain possessed entire gadB-gadC genes (1380 and 1434 bp, respectively) in its genome (Fig. 3). The translational product (459 amino acid protein) of the gadB gene was identical to that of strain APC151 with known GABA synthesis ability. The survey of 65 publicly available complete genome sequences of S. thermophilus found the presence of the genes in only 18 strains, and the genetic organizations around the GAD systems presented a high variability for the same species (Fig. 3, Supplementary  Fig. 5). Interestingly, in EG007, the gadB-gadC genes were flanked by multiple transposases elements (three IS6 family transposase ISLmo4 and an ISL3 family transposase IS1193), and the genetic organization was closest to the strain EU01 among the 18 strains. Similarity searches of the transposase elements against ISFinder DB showed near identity with the known insertion sequences of Listeria monocytogenes, Enterococcus faecium, and Lactococcus lactis (Fig. 3). It implies that an ancestral strain of EG007 may have originally acquired the operon by HGT from other GABA-producing microorganisms. According to previous findings, it has been reported that strains from L. monocytogenes, L. lactis, E. faecium were capable of producing GABA [72][73][74] . Furthermore, since microorganisms are able to raise the cytoplasm and extracellular pH by exporting the produced GABA, it is speculated that having the GAD system can be also beneficial for the microbe itself, by allowing them to survive better under acidic conditions 75 .
GABA plays an important part in mood and stress response by acting as a major inhibitory neurotransmitter in the mammalian central nervous system [76][77][78] . In addition to that, recent researches have assessed its essential role in cognitive functioning in animals and humans. For instance, Auger et al. revealed that the prefrontal GABA hypofunction severely disturbed spatial reference and short-term memory in rats 79 , and in humans, it was found that the GABAergic levels in the dorsal anterior cingulate cortex were significantly decreased in patients with schizophrenia and psychotic disorder 80 . In studies using rats, oral intake of GABA increased hippocampal Scientific Reports | (2022) 12:13274 | https://doi.org/10.1038/s41598-022-14837-z www.nature.com/scientificreports/ GABA level and enhanced learning and memory performances, which was confirmed by measuring NOR, maze, and PA test 81,82 . These are in agreement with the present results that the indices of spatial (Y-maze SA) and nonspatial working memory (NOR) processes were improved following supplementation of EG007. Moreover, in recent studies by Yunes et al. and Patterson et al., it was demonstrated that ingestion of GABA-producing LABs had positive effects on depressive-like behavior in the mice model 76,78 . As shown in previous studies, GABA receptors present on the enteric neurons of the GI tract, and the expression of the receptors are increased upon GABA-producing probiotics consumption. It is assumed that the GABA produced in the GI tract is able to directly modulate the host GABAergic signaling process using the enteric GABA receptors and vagus nerve as a route of communication 17,[83][84][85] . We, thus, presumed that EG007 could have direct effects on the cognitive ability enhancement of the mice through influencing the CNS via the gut-brain axis. In summary, EG007 possessed genes encoding various bioactive molecules beneficial for modulating host health in many aspects, and by screening antibiotic resistance and virulence-related genes, we also concluded that it can be considered safe as none of those genes were detected in the genome. We found evidences in the genome of EG007 that the ancestral strain might have undergone HGT to obtain genes related to the EPS, bacteriocin, and GABA productions which could also increase fitness in the GI tract. These acquisitions, consequently, might contribute to enhance probiotic functionalities. Especially, as one of the rare GAD system-possessing strains among S. thermophilus 86 , EG007 can be a great candidate to serve as next-generation probiotics with possible gut-brain axis modulating capability. Similar to the preceding studies, GABA produced by EG007 might have directly promoted the cognitive ability of ST supplemented mice group by stimulating enteric GABA receptors. Nanopore sequencing of 16S-23S operon and identification of microbial alterations of EG007 treated mice. To investigate whether EG007-related alteration in the intestinal microbiome was a component of memory enhancement, we compared and analyzed the gut microbial communities of EG007 and control groups. In general, for taxonomic classification of the microbial community, only a few hypervariable regions of the 16S rRNA gene have been utilized as a taxonomic marker, but it is known that these limited regions classify only to family-or genus-level and gives varied results depends on the region selections [41][42][43][44][45] . To take advantage of long-read sequencing technologies, an increasing number of studies are attempting to apply longer regions of markers such as full-length 16S and 16S-23S rRNA which allow higher and more accurate resolution of the microbiota [45][46][47][48] . In this study, the 16S-23S rRNA region (approximately 4200 bp) of the ribosomal operon was chosen as the marker to obtain increased resolution at the species level 45 . We randomly collected eight fecal sam-    (Table 3). We were able to detect a total of 1090 taxonomic units at the species level, with an average of 607 species and 631 species were detected for the control and ST group, respectively. In beta diversity analysis measured by PCoA for both weighted and unweighted UniFrac metrics and nMDS based on Bray-Curtis dissimilarity, subtle separation by groups was observed, even though Permutational Multivariate Analysis of Variance (PERMANOVA) failed to detect a significant difference ( Supplementary Fig. 6). Likewise, alphadiversity was not significantly affected by EG007 supplementation as estimated by several indices (Chao-1, ACE, Observed, Simpson's, and Shannon's). The composition of gut microbial communities at hierarchical taxonomy levels from the two groups were presented in Supplementary Fig. 7. The predominant phyla were Bacteroidetes, Firmicutes, and Actinobacteria, accounting for more than 95% together. At the genus level, Muribaculum was the most abundant genus, followed by Lactobacillus, Bacteroides, and Alistipes, similar to the previous mouse gut microbiota analysis [87][88][89] , and 163 common genera were characterized.
We further performed differentially abundant microbiota analysis using TMM-normalized GLM method in EdgeR package to identify microbial alterations that significantly contributed to differences between groups. We identified 25 taxonomic units across all taxonomic levels that were significantly over-or under-represented by consumption of EG007 (P-value < 0.05) (Fig. 4). After consumption of EG007, 11 significantly increased in abundance, and significantly lower levels of 14 taxonomic units were observed (Fig. 4). The altered species were  Table 3. Summary of taxonomic assignment results of reads generated using 16S-23S operon sequencing.  www.nature.com/scientificreports/ Phascolarctobacterium faecium that increased about eightfold in ST group mice were previously reported to be positively associated with the positive mood in the human host 99,100 , and beneficial effects of P. faecium also have been identified in several studies 101,102 . In addition, EG007 supplementation led to significant increases in the proportion of Ruminococcus species. Notably, prior studies have documented the association of genus Ruminococcus to AD, cognitive impairment, and depression 100,103,104 . Taken together, the results imply that supplementation of S. thermophilus led to positive changes of gut microbiota which may contributed to the beneficial effects in improvement of cognitive ability in mice. A recent study evaluating changes in the composition of the human gut microbiome upon yogurt consumption also observed significant increases in S. thermophilus and Ruminococcus species 105 . While a mouse microbiome catalog revealed that the human and mouse microbiota share a considerable number of microbial genera and showed significant overlap of annotated functions, it should be noted that there still exist large differences in the quantitative and qualitative representation of taxa between the two species 89,106 . Moreover, there are differences in microbial composition between mouse strains, and even mouse and human strains of the same microbial species can be very different 107 . Therefore, there are limitations to predicting responses in humans from such results in animal models. Further clinical studies involving human subjects are needed to evaluate whether the EG007 administration can induce similar alteration in the human gut microbiome and their cognitive functions.

Associations between gut microbiota and cognition traits.
In order to identify bacterial communities related to cognitive abilities, we further conducted correlation analysis between the normalized abundance of each taxonomic unit and the two improved cognition traits (Y-maze SA and NOR) via Pearson correlation analysis. 44 taxonomic units including 26 at the species level were significantly associated with spatial (Y-maze SA) and non-spatial (NOR) recognition memory (P-value < 0.05) (Supplementary Table 2). The differential ratio in the NOR test was correlated with 22 taxonomic units, including 15 positively correlated and 7 negatively correlated. The alteration score in the Y-maze test was correlated with 22 taxonomic units, including 13 positively correlated and 9 negatively correlated. Among them, Pediococcus inopinatus (r = 0.90) and S. salivarius (r = 0.89) had the most highly positive correlation with the differential ratio and the alteration score, respectively. It is worth noting that the well-known lactic acid bacteria, Lactobacillus fermentum (r = 0.73) and P. inopinatus displayed a significantly positive correlation with the non-spatial recognition memory, and a recent study indicated that a neuroinflammation inhibitory effects of P. inopinatus on glial inflammation and ameliorating ataxia symptoms of Parkinson's disease mouse model 108 . P. inopinatus was also positively correlated with the abundance of S. thermophilus (r = 0.79). Interestingly, the abundance of Staphylococcus aureus, generally known as an opportunistic pathogen, was significantly negatively associated with the alteration score (r = − 0.78) while other Staphylococcus species (S. schleiferi and S. pseudintermedius) displayed the opposite relationship (r = 0.72, r = 0.73, respectively).
Among the 44 microbiota detected to have correlation with the cognition traits, we observed that 8 were also significantly altered in the ST group, indicating these taxonomic units were not only modulated by EG007 supplementation but also closely related to the cognitive ability (Fig. 5). As shown in Fig. 5, three (S. thermophilus, genus Streptococcus, and family Streptococcaceae) were associated with the non-spatial recognition memory, and eight (Ruminococcus albus, Ruminococcus bicirculans, Ruminococcus champanellensis, genus Bittarella, S. salivarius, S. streptococcus, genus Streptococcus, and family Streptococcaceae) were associated with spatial recognition memory. Both of the scores exhibited a positive association with the abundance of S. thermophilus and its higher taxonomic levels. The results showed that three Ruminococcus species, whose abundances were significantly enriched by EG007, were positively correlated with spatial recognition memory. Ruminococcus species are generally described as consistently present SCFA-producing bacteria in the healthy human gut 90,109 , and according to previous researches, the relationship between the genus Ruminococcus and the brain functions has been described vary widely. Some studies suggested elevated fecal Ruminococcus levels in children or mouse model with ASD [110][111][112] , while others observed decreased fecal Ruminococcus in ASD children compared to the healthy controls 113,114 . An increase of genus Ruminococcus was also shown upon treating antidepressants or probiotics on the stress-induced mice 115 , and the negative association with the amyotrophic lateral sclerosis patients was reported by another gut microbiome study 116 . Contrastingly, another report found that Ruminococcus flavienciens was able to diminish the effects of antidepressants on the depressive-like behavior in mice 117 . These inconsistent results could be attributed by the differential influences of Ruminococcus species, thus the species level investigation is suggested to more clearly understand the association.
In this study, R. albus was increased 2.93-fold in mice supplemented with EG007 and had a significantly positive correlation with the spatial learning and memory (r = 0.63), and it was previously demonstrated that intestinal R. albus had neuroprotective effects by reducing oxidative stress and increasing BDNF level in brain 118 . Also, the results are paralleled by the finding that R. albus was not found in the stools of children with ASD, while a significant abundance of R. albus was found in control children 110 , suggesting its positive role. Any findings regarding R. bicirculans (increased 2.99-fold, r = 0.75) and R. champanellensis (increased 2.90-fold, r = 0.75) have not previously been reported so far. In addition to exerting various health benefits in the colon and the peripheral tissues, SCFAs play a pivotal role in the MGB crosstalk through crossing the blood-brain-barrier (BBB) and influencing the enteric nervous system activity 119 . Ruminococcus genus has been identified as one of the bacterial communities responsible for intestinal production of SCFAs 109 . SCFAs mainly consist of butyrate, propionate, and acetate 120 , and it has been suggested that each of them exerted a different function on the nervous system physiology. While butyrate is known as involved in histone deacetylase inhibition, anti-depressant effects, and regulating BBB and gut permeability 109,121 , propionate was reported to cause developmental delay or seizures 122 and alter dopamine, serotonin, and glutamate systems in a manner similar to that observed in ASD 123,124 . Previous studies have also found that a subset of ASD patients has high levels of propionate-producing species 114 127 , and the acetate level was significantly downregulated in the AD model 128 . Thus, it is speculated that the different fermentation products of Ruminococcus species potentially resulted in the different associational pattern as R. albus and R. bicirculans are known to produce acetate and formate from carbohydrates 118,129 , whereas other species such as Ruminococcus obeum and Ruminococcus bromii were reported to associate with propionate production 130,131 . Future biochemical studies are required to provide further insights into the underlying molecular mechanisms through which these species contribute to the brain function, and also cognition-associated microbiota affected by EG007 supplementation presented in this study will serve as preliminary data for future associative studies.

Conclusion
In summary, our study observed that 8-weeks consumption of EG007 markedly promoted short-term learning and memory skills in healthy young male mice. By constructing complete genome sequence of EG007, we identified genes encoding various metabolites that might have contributed to the enhancements in gut microbiota and brain function, including bacteriocin-coding genes and the genes encoding glutamate decarboxylase (gadB) and glutamate/GABA antiporter (gadC), which are necessary for GABA production. Further, as a result of gut microbiota community analysis utilizing 16S-23S sequencing, we gained insights into EG007-mediated compositional changes at the species level. Key signature alterations were observed including down-regulation of inflammation or infection-related species and enrichment of species, previously reported to have an association with the CNS functions such as P. faecium and Ruminococcus species. Moreover, correlation analysis highlighted that eight significantly modulated microbiota upon EG007 supplementation were also closely related to the cognition traits, which have not been reported so far. Collectively, the results above suggested that S. thermophilus EG007 was an important exogenous factor that can improve cognitive functions. Although we obtained meaningful results, there are still several limitations that needed to be mentioned. For behavior tests, the forced alternation test could be performed before the spontaneous alternation test. Although there were two weeks of gap between the two tests, measuring the natural curiosity against the novel arm would be more accurate if the mice did not have any prior information about the space. Also, this study does not provide direct evidence of memory improvement linking the MGB axis. Therefore, further biochemical studies identifying gut-brain related biomarkers are needed to elucidate the mechanisms of action underlying the cognition-enhancing effects of EG007 or the microbiota modulated by EG007.
Despite these limitations, this is the first study examining the effects of a single S. thermophilus strain supplementation on brain function and also one of few studies focused on the effects in young healthy subjects, rather than populations with neurological disorders or age-related cognitive declines. Besides, in the present study, nanopore sequencing was employed in the genomics and metagenomics analysis to investigate the probiotics and psychobiotics properties. This application of long-read sequencing technology had a critical role particularly in providing more accurate and higher resolution of taxonomic inferences of healthy gut microbiota which have more complex characteristics, thereby revealing more detailed S. thermophilus-derived changes and microbiotacognition relationships at the species level. The genomic features and gut microbial modulations revealed in this study will serve as preliminary data for future associative studies and human clinical trials and will provide a potential of S. thermophilus in assisting cognitive function enhancements by possible MGB axis modulating capability, which can be applied for the more general population of healthy and young people.

Animals and housing. All animal experiments were performed following NIH Guide for the Care and
Use of Laboratory Animals approved by the Institutional Animal Care and Use Committee of Seoul National University (approval number: SNU-190607-4-3). The study is reported in accordance with ARRIVE guidelines. Male SPF C57BL/C mice (n = 36, 4 weeks old on arrival) were purchased from the Youngbio (Seongnam, Republic of Korea). Mice were housed in groups of four littermates per cage (33 cm × 15 cm × 13 cm, L × H × W) in the animal facility at the Seoul National University and allowed to acclimatize for one week prior to dietary administration. Cages of mice were randomly divided into three groups (ST, LP, and Control, n = 11-12/group). All mice were provided with standard chow diet in pelleted form, and the groups were provided with sterilized water (Control group) or sterilized water supplemented with 1.08 × 10 10 CFU of S. thermophilus EG007 (ST group) or 3.12 × 10 10 CFU of L. plantarum A003F7 (LP group) ad libitum throughout the whole study. They were maintained in a ventilated room under standard controlled laboratory conditions: a 12-h light/dark cycle (lights on at 7:00 am), a temperature of 22 ± 2 °C with 50 ± 5% humidity. Cages were changed once a week by the same experimenter. Fig. 1. After a week of acclimatization to the animal facility, 5-weeks-old mice of experimental groups (ST; n = 12 and LP; n = 11) were provided EG007 or A003F7, respectively, in drinking water daily ad libitum for 8 weeks, and control group (control; n = 12) were given normal drinking water ad libitum throughout the study. All mice were weighed once weekly and water consumption was monitored every day. From week 5 (9 weeks of age) onward, all mice were subjected to the same series of behavior tests to assess their cognitive performance, the Y-maze test (spontaneous alternation and forced alternation) for short-term spatial memory, the novel object recognition test for short-term nonspatial memory, and the passive avoidance test for long-term associative memory. Tests were ordered from the least to the most stressful task, including resting days between tests, to avoid carryover effects. All behavior tests were performed between 1 and 6 pm in the active phase of the animal under dim red light to reduce anxiety in the animals. On the day of testing, mice were transferred in their original cages to the behavior testing room 2 h before the test to minimize stress. Animals were tested one at a time in a counterbalanced fashion regarding testing order and under the same conditions, and all apparatus used in the testing were thoroughly cleaned with 70% EtOH between animals or sessions to remove odors. Test sessions were recorded using camera mounted facing straight down, and two independent experimenters blinded to conditions manually scored behaviors of the videos. All mice were sacrificed 5 days following the last test (week 9).

Study design. The graphical experiment design is presented in
Probiotic administration. Both strains (EG007 and A003F7) were isolated from fermented dairy products in the present laboratory. In this study, to avoid stress in the animals caused by daily use of oral gavage, mice were administered probiotics, dissolved in drinking water. The water containing EG007 or A003F7 was freshly prepared just before administration from bacteria grown overnight and changed every morning on a daily basis www.nature.com/scientificreports/ throughout the study (8 weeks). For the control group, sterile water was provided and changed every morning as well. Each probiotic strain was cultured aerobically in de Man, Rogosa, Sharpe (MRS) media at 37 °C for 9 h each, harvested by centrifugation at 4000 rpm for 10 min, and washed twice with sterile saline. The collected cells were resuspended in the same volume of sterile water at an average concentration of 1.45 × 10 9 CFU/ml (EG007) and 3.73 × 10 9 CFU/ml (A003F7). Water intakes were recorded every day throughout the experiment. The viability of probiotics and the final dose in the drinking water have been confirmed by plating resuspended water on the MRS plates every day. The final concentration ingested by each mouse was 1.08 × 10 10 CFU (EG007) and 3.12 × 10 10 CFU (A003F7) per day based on plating results ( Supplementary Fig. 1).

Y-maze test. Two versions of Y-maze tests were performed: Spontaneous Alternation test and Forced
Alternation test. The Y-maze apparatus (Jeung Do Bio & Plant CO., LTD, Seoul, Korea) was consisted of three Y-shaped white plastic arms symmetrically disposed at 120° angles from each other (40 cm long, 5 cm wide, and 10 cm high) and used in both tests. To differentiate three arms, ground in the testing room around each arm was marked, and they were kept in place throughout the testing period. Y-maze test is generally regarded as a measure to evaluate short-term spatial working memory in mice 132,133 based on their innate tendency to explore novel space, and it reflects the functions of prefrontal and hippocampal systems 134 . For Spontaneous Alternation, mice were randomly placed at the end of one of the arms to avoid placement bias, facing the wall, and allowed to freely explore the three arms for 6 min. The sequence of arm entries and the total number of arm entries were recorded to measure the alternation score. A spontaneous alternation is defined as entering a different arm in three consecutive entries (e.g., ABC, CBA, or BCA but not BAB), and the alternation score was calculated as follows: Alternation score (%) = (number of alternations) (total number of arm entries−2) × 100 . The total number of arm entries was also used as a locomotor activity indicator.
Forced Alternation test consisted of the training phase, intertrial interval, and testing phase 135 . During the training phase, access to one of the arms (novel arm) was blocked by a white barrier, and mice were placed in the start arm and allowed to explore the two open arms of the maze (start and familiar arm) for 5 min. After 1 h of an intertrial interval, the testing phase was performed with the barrier removed this time. The mice were again placed in the Y-maze in the same start arm, and all three arms (start, familiar, and novel arms) are opened to explore for 5 min. During the testing phase, the time taken for mice to enter the novel arm (latency), the percentage of time spent in the novel arm, and the percentage of entries into the novel arm were calculated to determine the tendency to explore the novel arm. For both of the tests, arm entries were counted when all four paws entered into the 1/3 point of the arm, and if a mouse climbed the maze wall, it was immediately returned to the abandoned spot.
Novel object recognition test. Novel object recognition (NOR) test was performed to evaluate shortterm non-spatial memory of mice, exploiting their innate tendency to explore a novel object 136 . It reflects the dorsal hippocampal function which is associated to the ability to recognize a familiar object 137 . This test consisted of three phases: training phase, intertrial interval, and testing phase. Prior to the test, several objects were tested to avoid object bias, and objects with equal preference were selected for the NOR test. The objects used were of different shape and color but of equivalent texture and volume (size 2-3 × 4-5 cm): two star-shaped purple plastic toys and two triangle-shaped green plastic toys. The object recognition testing arena (Jeung Do Bio & Plant CO., LTD, Seoul, Korea) was made of a white plastic open field (40 cm × 40 cm × 40 cm, L × W × H). During the training phase, two identical objects were placed in opposite quadrants of the testing arena (e.g., one in the NW corner and one in the SE corner), and mice were placed facing the wall in the corner, equidistant from both objects and allowed 10 min for free exploration. After 1 h of an intertrial interval, in the testing phase, mice were positioned again in the same location of the arena, facing the wall, in which one of the two identical objects was substituted with a novel object. For each mouse, objects were placed on the same diagonal location as used in the training phase, and mice were given 5 min to freely explore the objects. The diagonal location and the position of the novel object (left or right) was randomly assigned and counterbalanced between each mouse and conditional group. Preference for a novel over a familiar object was determined by measuring the amount of time explored the novel object relative to the familiar one, and the results were expressed as a discrimination ratio. The discrimination ratio was calculated as the percentage of novel object recognition time as follows: Discrimination ratio = Time novel Time novel +Time familiar × 100 . Exploratory behavior was counted when a mouse approached an object with its nose pointed towards the object, with active sniffing or vibrissae sweeping within 2.5 cm of the object. A mouse climbing on top of the object or approaches without paying attention was not considered.
Passive avoidance test. The passive avoidance (PA) test was performed to evaluate long-term associative learning and memory 138 of mice through Pavlovian fear conditioning, based on the innate aversion to the brightly illuminated areas. We used a step-through shuttle-box (Jeung Do Bio & Plant CO., LTD, Seoul, Korea) consisted of two chambers (white and black chambers) separated by a sliding door, and the floor of the chambers consisted of stainless-steel rods (2 mm diameter) spaced 1-cm apart. The white chamber (21 × 21 × 30 cm) was illuminated, while the black (21 × 21 × 30 cm) remained dark. During the training phase, mice were placed in the illuminated chamber with the door closed, and the door was raised noiselessly when the mice were facing away from it. When the mice entered the dark chamber, the door was closed and a mild foot shock of 0.25 mA was delivered for 3 s through the stainless-steel rods using a shock generator. Mice were remained in the dark chamber for 30 s after shock, and then returned to the home cage. During the process, mice learned the associative rule that a black chamber is equal to shock 139 . 24 h post training, the testing phase commenced. Trained mice were again placed in the illuminated chamber, and the same procedure was conducted except for the foot shock. In both phases, the latency time to entering the dark chamber with all four paws was measured with a cut-off Microbial community analysis and statistical analysis. After the eight sequencing runs were completed, fast5 files were base-called using Guppy GPU basecaller (v4.2.2), and Porechop (v0.2.4, https:// github. com/ w1bw/ Porec hop) was used to remove the sequencing artifacts and chimeric reads. After the trimming step, reads were filtered by size to retain sequences with 3500 to 5000 bp length. To assign taxonomy, the processed reads from each sample were aligned against MIrROR database (mirror.snu.ac.kr; https:// github. com/ seoldh/ MIrROR) which was constructed in our accompanying study using Minimap2 aligner (v2.17) 140 with '-secondary = no' option. The MIrROR database contains sequences of 97,781 full 16S-23S rRNA operon sequences of the 43,653 bacterial strains representing 9485 species, annotated according to GTDB taxonomy. We kept only those reads that aligned to the reference with a block ≥ 2500 bp and the number of matching bases ≥ 1500 bp. For reads that hit more than one reference sequence, only the alignments with the highest alignment score (AS score; Smith-Waterman alignment score) were retained. Bacterial diversity was calculated by alpha-diversity (Shannon's index, Simpson index, Observed OTUs, ACE) and beta-diversity (Weighted and Unweighted Unifrac and Bray-Curtis dissimilarity). For detection of differentially abundant microbiota, a negative binomial based generalized linear model (GLM) was employed using EdgeR R package 141 with trimmed mean of M-value (TMM) normalization method for consideration of different read production.
DNA extraction and whole genome sequencing of S. thermophilus. EG007 was grown aerobically in MRS broth for 9 h at 37 °C. Genomic DNA was extracted using PureHelix™ Genomic DNA Prep Kit (Solution Type)-Bacteria (NanoHelix, Daejeon, Korea), and isolated gDNA was quantified and qualified using gel electrophoresis, 260/280 nm absorbance ratio and Quant-iT™ PicoGreen™ dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). The library was prepared using the ONT 1D ligation Sequencing kit (SQK-LSK109 Genome assembly, annotation, and analysis. Base-calling was carried out using Guppy GPU basecaller (v4.2.2). Sequencing artifacts and chimeric reads were removed using Porechop (v0.2.4, https:// github. com/ w1bw/ Porec hop), and reads with a quality score below 10 were filtered out using Nanofilt (v2.7.1) 142 . Genome assembly was conducted using CANU (v2.1.1) 143 with genomeSize = 1.9 Mb parameter. In order to increase consensus accuracy, multiple rounds of polishing with racon (v1.4.17) 144 and medaka (v1.0.3) (https:// github. com/ nanop orete ch/ medaka) were conducted until no additional correction could be made. Finally, the assembled reads were circularized using Circlator 145 . Genome annotation was performed using the Prokka (v1.14.6) with-rfam option to enable a search for ncRNA, and automatic annotation results were also collected from Rapid Annotation using Subsystem Technology (RAST). The predicted CDSs were then assigned in different clusters of orthologous group (COG) functional categories using eggnog-mapper tool (v2.0.1b) 146 , and COGs were retrieved using a bash script-eggnog-mapper_COGextraction (https:// github. com/ raymo ndkiu/ eggnog-mapper_ COGex tract ion). Genomic Islands (GIs) were detected through the IslandViewer4 web-based resource 147 , and the CRISPRs were identified with CRISPRCasFinder web tool 148 . For putative prophage detection, PHASTER was utilized 147 . Antibiotic resistance genes and virulence factor-related genes were predicted using CARD 149 , ResFinder 150 , and VFDB 151 . Lastly, ISFinder 152 was used to identify insertion sequences, and BAGEL4 153 was utilized to identify potentiality to produce bacteriocins. For identification of stress tolerance related genes, sequence information for different probiotic genes 154 were obtained from the NCBI database and used for BLAST-based sequence similarity search. www.nature.com/scientificreports/ Phylogenetic analysis. The phylogenetic tree was constructed with 849 1-to-1 orthologous genes in 65 publicly available complete genomes of S. thermophilus and EG007 identified by PorthoMCL 155 . The genes were individually aligned using MUSCLE 156 and refined using Gblock 157 . Concatenated sequences were subjected to construct a neighbor-joining tree with 1000 bootstrap replicates using MEGA-X 158 . Streptococcus salivarius L25 genome was used as an outgroup.

Data availability
The datasets generated during the current study are available in the NCBI BioProject accession no. PRJNA698776 and PRJNA834938.